Process for the preparation of aspartic acid

ABSTRACT

The invention provides a process for the preparation of aspartic acid via a fermentation process for the preparation of ammonium fumarate, wherein the pH of the fermentation broth is controlled by the addition of a calcium base to produce a calcium fumarate precipitate, characterized in that ammonium fumarate is produced by separating the precipitated calcium fumarate from the fermentation broth, and reacting the same with a reagent selected from ammonia, ammonium carbonate, ammonia in combination with CO 2  and mixtures thereof, to form ammonium fumarate and a co-product selected from calcium carbonate and calcium hydroxide, wherein the energy of indirect neutralization of fumaric acid by ammonia serves as the driving force for the conversion of calcium fumarate to the desired ammonium fumarate product and for the regeneration of a calcium base reagent, and wherein diammonium fumarate is enzymatically converted to ammonium aspartate and acidulated to from aspartic acid.

The present invention relates to a process for the production ofaspartic acid.

More particularly, the present invention relates to the preparation ofaspartic acid via a fermentation process for the preparation of ammoniumfumarate.

Aspartic acid is the 2-aminobutanedioic acid, occurring in its L-form inanimals and plants. It is a reagent in the production of the artificialsweetener aspartame. L-Aspartic acid is one of the amino acids that isdifficult to produce directly by fermentation. Consequently it isproduced enzymatically by conversion of diammonium fumarate to ammoniumaspartate which is then acidulated.

GB 1 004 218 discloses a process for the production of L-aspartic acid,comprising mixing a strain of Pseudomonas trifolii with an aqueoussolution of fumaric acid or a fumarate, and ammonia or an ammonium salt,allowing the mixture to stand under neutral or mildly alkalineconditions to allow L-aspartic acid to accumulate therein, andrecovering. the L-aspartic acid from the mixture. Preferably diammoniumfumarate serves as both the fumarate and the ammonium salt.

Diammonium fumarate is obtained by neutralizing fumaric acid withammonia. About one half of the total aspartic acid production cost isdue to the fumaric acid consumed. Fumaric acid is presently produced bycatalytical isomerisation of maleic anhydride or maleic acid producedmostly from benzene. In the early 1940's fumaric acid was made on acommercial scale by Pfizer by fermentation of glucose using a strain ofthe fungus Rhizopus. Production was stopped when the economically moreattractive synthesis from maleic acid was developed.

Since then many improvements to the fermentation were made. U.S. Pat.No. 4,877,731 describes an improved fermentation process for producingcarboxylic acids. The improvement comprises growing fungi of the genusRhizopus in a culture medium under conditions of controlled oxygenavailability. Goldberg and Steiglitz (Biotechnology and BioengineeringVol. 27, 1067-1069 (1985) have found that adding surfactants orvegetable oils increased the rate of fumaric acid accumulation. Otherimprovements and optimizations were made by Rhodes et. al (AppliedMicrobiology, 7, 74-80 (1959)), Moresi et. al (Applied Microbiology andBiotechnology, 36, 35-39, 1991), Kautola and Linko (Applied Microbiologyand Biotechnology, 31, 448-482 (1989)) and others.

In spite of these improvements fumaric acid is not produced presently byfermentation. This is at least partially due to the complicated andcostly process for its recovery from the fermentation broth. In thefermentation, as the pH of the broth drops in consequence of production,the rate of fumaric acid production slows down and eventually ceases.CaCO₃ is added as a neutralizing agent to prevent this self-inhibition.Because of its insolubility, CaCO₃ offers the advantage of all-at-onceaddition and therefore eliminates the requirement of a control systemfor base addition. However, because of the low solubility of calciumfumarate at the fermentation temperature, the product precipitates outof the broth throughout the fermentation. At the end of thefermentation. the broth, containing a slurry of calcium fumarate. isheated to 160° C. in a reactor and is acidified to pH 1.0 by H₂ SO₄. Atthis temperature, both calcium fumarate and fumaric acid are soluble,but calcium sulfate precipitates out of solution and is filtered off.Fumaric acid crystals are recovered by cooling the filtrate.

The overall process thus consumes CaCO₃ and H₂ SO₄ as reagents producesgypsum as an undesired by-product, consumes energy for the reaction athigh temperature and requires handling an acidic solution at very hightemperatures. All these elements add to the cost of fumaric acidproduction through fermentation and render fumaric acid, produced byfermentation, an unfavorable reagent for aspartic acid manufacture.

Conversion of fumaric acid fermentation to aspartic acid fermentation bythe combination of Rhizopus and bacteria (Proteus vulgaris) wasattempted, but the results were not favorable (Hotta and Takao, HakkoKogaku Zasshi 51, 12-18 (1973)). An alternative could have beenneutralization of the fermentation by ammonia to directly form ammoniumfumarate for further purification or direct conversion to aspartic acid.That is however not attainable as explained by Goldberg and Steigliz:"The key point in fumaric acid accumulation is adding a limiting amountof nitrogen (e.g. urea) as cells starved for nitrogen produce fumaricacid and not biomass from glucose." Another suggestion was to use Na₂CO₃ as the neutralizing base to form sodium fumarate in the fermentationbroth and recovering the fumaric acid therefrom by acidulation (Ganglet. al, Applied Biochemistry and Biotechnology 24/25, 663-677, (1990)).However formation of a soluble fumarate salt in the fermentation liquorhas a negative effect on production.

With this state of the art in mind, there is now provided, according tothe present invention, a process for the preparation of aspartic acidvia a fermentation process for the preparation of ammonium fumarate,wherein the pH of the fermentation broth is controlled by the additionof a calcium base to produce a calcium fumarate precipitate,characterized in that ammonium fumarate is produced by separating saidprecipitated calcium fumarate from said fermentation broth, and reactingthe same with a reagent selected from ammonia, ammonium carbonate,ammonia in combination with CO₂ and mixtures thereof, to form ammoniumfumarate and a co-product selected from calcium carbonate and calciumhydroxide, wherein the energy of indirect neutralization of fumaric acidby ammonia serves as the driving force for the conversion of calciumfumarate to the desired ammonium fumarate product and the regenerationof a calcium base reagent, and diammonium fumarate is enzymaticallyconverted to ammonium aspartate and acidulated to form aspartic acid.

In preferred embodiments of the present invention said calcium baseco-product is recycled to the fermentation broth.

Preferably said precipitated calcium fumarate from said fermentationbroth is subjected to purification before reaction with said reagent.

In especially preferred embodiments, said precipitated calcium fumarateis recrystallized before said reaction.

U.S. Pat. No. 5,352,825 teaches a method for the recovery of organicacid salt from an impure process stream, comprising the steps of:

a. obtaining a solution of an organic acid salt of interest,concentrated to within about ten percent of the saturation point;

b. adding a sufficient amount of crystallizing base to the concentratedsolution of the organic acid salt to produce crystals of the salt; and

(c) separating the crystallized organic acid salt from mother liquor.The "crystallizing base" added in step b causes crystallization of theorganic acid salt present already in the impure stream or producedtherein by adding a "neutralizing base". The "crystallizing base" neednot be consistent with that of the "neutralizing base". Thus, Example 3of the patent describes production of trisodium citrate from a solutioncontaining it by adding ammonia as a crystallizing base.

In contrast to U.S. Pat. No. 5,352,825, in the new process the addedreagent does not facilitate the production of the calcium fumaratepresent in the fermentation broth, but causes its conversion to ammoniumfumarate.

Thomsen (U.S. Pat. No. 3,030,276) describes the manufacture of fumaricacid from lignified cellulose which comprises; digesting said lignifiedcellulose with a solution of caustic soda under the conventionallimitations as to alkali concentration, time and. temperature, untilsubstantially all the non-cellulose portion shall have been dissolved;separating the spent cooking liquor for subsequent conventionalregeneration and recycling to said digestion step from the celluloseresidual and saccharifying the latter with dilute sulphuric acid underthe conventional limitations as-to acid concentration, time andtemperature; commingling the resultant sugar solution with arecirculated excess of calcium carbonate produced in a subsequent stepand with a micro-organism suitable for the conversion of sugar intofumaric acid; commingling the resultant mixture of calcium sulphate,fumarate and carbonate with sufficient carbonated ammonia to decomposeall such sulphate and fumarate forming calcium carbonate and sulphateand fumarate of ammonium; separating and recycling such calciumcarbonate and evaporating the resultant solution to substantialsaturation and acidifying said solution of ammonium sulphate andammonium fumarate with the stoichiometric amount of sulphuric acid todecompose all resident fumarate and crystallizing out the liberatedfumaric acid from the residual solution of ammonium sulphate.

While a conversion of calcium fumarate and cabonated ammonia to ammoniumfumarate and calcium carbonate is described in said patent, it does notteach the economic formation of ammonium fumarate for aspartic acidproduction.

Firstly, according to Thomsen the production of fumaric acid as the soleproduct is not economic and should be combined with the production ofurea. "Owing to its cost, fumaric acid is as yet of little importance .. . Urea and its production is too well known . . . but it is the aimand object of my process to link the simultaneous production of both insuch a manner that the discard from one series of steps supplements theneed of the other series, eliminating all waste effort and cheapeningthe cost of both. The best way to evaluate said linkages . . . thechemical reactions involved are old, only the combinations being new.That, however, is the important part for it is upon such that theeconomy, hence the value of my process rests."

Secondly, the conversion of calcium fumarate to calcium carbonate andammonium fumarate is made in U.S. Pat. No. 3,030,276, in the presence ofgypsum and ammonium sulfate "comingling the resultant mixture of calciumsulfate, fumarate and carbonate with sufficient carbonated ammonia todecompose all such sulfate and fumarate forming calcium carbonate andsulfate and fumarate of ammonium." Thomsen does not teach that efficientconversion can be achieved without the facilitating effect of saltingout by the presence of other salts. This could be of particularimportance in the current case where one of the reagents and one of theproducts are solids and the reaction kinetics could be slow. In fact, nodetails of the reaction conditions, such as temperature, pressure andwhat is "sufficient carbonated ammonia", are given and no claims aremade as to the degree of the conversion and as to the productconcentration. No examples are provided.

Furthermore, Thomsen does not teach the recovery of ammonium fumaratepure enough and concentrated enough for conversion into aspartic acid.In fact, his process requires concentration of the ammonium fumaratecontaining solution and purification through the addition of sulfuricacid and crystallization of fumaric acid "evaporating the resultantsolution to substantial saturation and acidifying said solution ofammonium sulfate and ammonium fumarate with the stoichiometric amount ofsulfuric acid to decompose all resident fumarate and crystallizing outthe liberated fumaric acid from the residual solution of ammoniumfumarate". One could indeed follow this process and react the separatedfumaric acid with ammonia to form ammonium fumarate for conversion toaspartic acid. It would, however, result in the production of at leastan equivalent amount of ammonium sulfate which is a waste or a low gradefertilizer.

Considering the proposed process for reacting calcium fumarate with areagent selected from ammonia, ammonium carbonate, ammonia incombination with CO₂ and mixtures thereof and based on the teachings ofthe prior art, one would expect several difficulties. The solubility ofcalcium fumarate at about ambient temperature, is low (2.11 w/v at 30°C.). Reactions of solid reagents are slow in many cases. An additionaldegree of complication is added by the fact that one of the reactionproducts, CaCO₃, is insoluble, which adds to the foreseen difficultiesin contacting the reagents for a rapid reaction. Another potentialdifficulty is the possibility of CaCO₃ precipitation on the surface ofcalcium fumarate crystalls, further slowing their interaction with thereagent.

The calcium fumarate reaction with sulfuric acid to form fumaric acidand gypsum seem to face a similar problem. There it was solved byconducting the reaction at 160° C., a temperature high enough tosolubilize the calcium fumarate. Adopting a similar solution to thepresent case could be difficult. Since ammonium carbonate decomposes atthese elevated temperatures, and since the solubility of ammonia, andparticularly that of CO₂ in the reaction medium decreases, the reactionrate is expected to slow. A possible solution is operating the reactionunder high pressure, but that would add significantly to the cost of theproduction.

Another element of high importance is that of pH adjustment to thecontradictory requirements of the reaction. The preferred product forthe conversion to aspartic acid is diammonium fumarate. This allowsoperating the reaction at high ammonium concentrations which facilitatesthe reaction. An excess of ammonia could be considered in order toassist in maintaining the CO₂ or CO₃ ²⁻ content of the reaction mixture.The possible difficulties are the need of removal of an excess ofammonia from the reaction mixture and the possible precipitation ofCa(OH)₂ rather than CaCO₃ as a by-product at high pH. The process ofthis invention could use Ca(OH)₂ as the neutralizing agent and reform itfrom calcium fumarate. CaCO₃ is, however, a preferred neutralizing agentin the fermentation as it introduces CO₂. Such CO₂ introduction isneeded to increase the fermentation yield.

Alternately one could design the process and the reagent ratio to formmonoammonium fumarate which would be converted later to aspartic acidafter the addition of ammonia. However, the dissociation constants offumaric acid should be noted: pKa₁ =3.03 and pKa₂ =4.44. The pH of areaction solution comprising mono ammonium fumarate would be lower than4. The dissociation constants of carbonic acid are high: pKa₁ =6.37 andpKa₂ =10.25. Calcium carbonate precipitation at pH of about 4 is verydifficult and even at the pH of diammonium fumarate, about 5, would notprecipitate easily high CO₂ pressure was found in many cases to assistin precipitation of bicarbonate salts (particularly NaHCO₃), but is notdesired in the present case.

Another aspect of importance is that of product concentration. Thisbioconversion should be fed with as concentrated as possible solution ofdiammonium fumarate for efficiency and for reducing losses in theaspartic acid recovery. According to the prior art one would expect thata significant amount of water would be needed in the reaction tomaintain a sufficient amount of calcium fumarate in solution. Thisamount of water would end up in the product ammonium fumarate and diluteit.

The fermentation of cabohydrates to fumaric acid (fumarate at thefermentation pH) is usually not very selective. Typically only about 80%of the total carboxylate formed is fumarate, the rest being typicallymaleate, succinate and alpha ketogluterate. In addition, thefermentation liquor contains other organic fermentation products such asglycerol. Those fermentation by-products, as well as other impurities,such as non-utilized carbohydrates, mineral cations and anions andnitrogen containing compounds, may cause difficulties in the next stepof bioconverting ammonium fumarate to ammonium aspartate. Even if not,these impurities could end up in the ammonium aspartate formed and causemajor difficulties in the recovery of pure aspartic acid at high yields.

With this background it was surprising to find that the reaction ofcalcium fumarate to convert it to ammonium fumarate and calciumcarbonate can be conducted under favorable conditions:

a. The amount of water needed in the reaction is limited and ammoniumfumarate can be obtained at a nearly saturated solution.

b. The reaction yield at about ambient temperature is nearly 100% andelevated temperatures are not required. In fact, high temperaturesreduced the reaction yield.

c. High CO₂ pressures are not required and practically full conversionis obtained at CO₂ pressure of less than 5 and preferably at a CO₂pressure of less than 2 atmospheres.

d. The calcium carbonate that forms does not coat the calcium fumarateand is obtained in clean, easy to separate crystals.

Thus it will be realized that in counterdistinction to Thomsen, thepresent invention does not require the production of fumaric acidthrough the wasteful addition of sulphuric acid, and instead is based onthe formation of ammonium fumarate. Most of the fermentation by-productsare removed in the process and the ammonium fumerate formed is pureenough for direct conversion to ammonium aspartate.

The reagent for the reaction is selected from ammonia, ammoniumcarbonate, ammonia in combination with CO₂ and mixtures thereof. Theamount of ammonia or ammonium ion in the reagent is preferably at leastone mole and more preferably about two moles per mole of calciumfumarate. Even more preferred is an excess of about 10% over the 2:1molar ratio since the bioconversion to aspartic acid is facilitated byan excess of ammonia. It was found that when the reagent is ammoniumcarbonate, the yield and the rate of the reaction are improved by addingto the reaction medium a small amount of ammonia to adjust the pH thereto about 10-11 prior to the addition of ammonium carbonate.

The amount of CO₂ or CO₃ ²⁻ in the reagent is preferably about one moleper mole of calcium fumarate. It could be introduced as a gaseous CO₂ oras ammonium carbonate or bicarbonate. In both cases the reaction ispreferrably performed in a closed system to avoid CO₂ losses. In casesof gaseous CO₂ addition the pressure in the reactor should stay below 5atmospheres.

Gaseous ammonia or aqueous ammonia or ammonium carbonate solutions canbe used. The amount of water introduced with the reagent is adjusted toa level that will not exceed the level equivalent to final fumarateconcentration of about 50% of saturation. Preferably the amount of wateris such that the final fumarate concentration is at least 80% ofsaturation.

The reaction temperature is preferably below 80°C. and even morepreferred below 50° C.

In a preferred embodiment the calcium fumarate is separated from thefermentation liquor, e.g., by filtration, and washed with water or withan aqueous solution from a previous step.

One can also take advantage of the low solubility of calcium fumarate atlow temperature and enhanced solubility at high temperatures. Thisprovides for a preliminary purification of the calcium fumarate formedin the fermentation by recrystallization. This recrystallization servesto remove the biomass and a significant amount of the impurities.

If needed, the ammonium fumarate formed in the reaction, can be furtherpurified by converting it back to water immiscible calcium fumarate(e.g. by reacting it with calcium hydroxide or carbonate) and washingaway the water soluble impurities.

Preferably the calcium base (carbonate or hydroxide) formed as aby-product is separated from the ammonium fumarate and reused as aneutralizing agent in carbohydrate fermentation to fumarate. Preferablythe calcium base is calcined prior to the reuse, whereby biomass left init is removed. In a most preferred embodiment the calcined calcium baseis quenched in water or in an aqueous solution and kept suspended in ituntil used. This suspension in water helps in removal of ashes left frombiomass burning and other ashes left from the fermentation step.

While the invention will now be described in connection with certainpreferred embodiments in the following examples so that aspects thereofmay be more fully understood and appreciated, it is not intended tolimit the invention to these particular embodiments. On the contrary, itis intended to cover all alternatives, modifications and equivalents asmay be included within the scope of the invention as defined by theappended claims. Thus, the following examples which include preferredembodiments will serve to illustrate the practice of this invention, itbeing understood that the particulars shown are by way of example andfor purposes of illustrative discussion of preferred embodiments of thepresent invention only and are presented in the cause of providing whatis believed to be the most useful and readily understood description offormulation procedures as well as of the principles and conceptualaspects of the invention.

EXAMPLE 1

15.4 g of solid calcium fumarate were mixed in a beaker of water and afew drops of 25% ammonia were added to adjust the pH of the solution toabout 10. Most of the calcium fumarate remained undissolved. 10.6 g ofsolid ammonium carbonate was added into the beaker that was held atambient temperature and was mixed strongly. After one hour of mixing,the beaker contained a large amount of solids. After filtration andwashing the solid was analyzed. The analysis showed that it was calciumcarbonate. The filtrate was analyzed for fumarate anion. The fumarateconcentration in it was 0.87 mol/kg (about 13% w/w of diammoniumfumarate), indicating a substantially complete conversion.

The filtrate was then converted enzymatically to ammonium asparate andthen acidulated with an equivalent amount of sulfuric acid toprecipitate out aspartic acid.

EXAMPLE 2

Solid ammonium carbonate was added gradually into 50 g mixed suspensioncontaining about 10% calcium fumarate until the carbonate to fumaratemolar ratio reached 1.1:1. The initial temperature of the suspension was22° C. After the addition of the carbonate the mixing was continued foradditional 30 minutes without any heating. Then the solids were filteredand washed, the filtrate was combined with the wash water and thecombined solution was analyzed for fumarate 99.5% conversion was found.

EXAMPLE 3

50 g. of a suspension containing about 10% calcium fumarate wereintroduced into a pressure vessel. Solid ammonium carbonate was added inone portion and the vessel was closed. The amount of ammonium carbonatewas adjusted to a carbonate fumarate molar ratio of 1.1:1. Strong mixingwas applied for 2 hours. No heating was applied. In the first 5-10minutes a pressure rise was observed. Then it decreased back to oneatmosphere. The pH of the final solution was about 8. Its fumaratecontent showed 95% conversion.

EXAMPLE 4

5 Kg. of fermentation broth analyzed and was found to contain: 329 g(2.84 mole) fumarate, 39.5 g (0.29 mole) maleate, 24 g (0.20 mole)succinate, 20.5 g (0.14 mole) alpha ketoglutarate and 76.5 g (0.83 mole)glycerol. The broth was filtered and the solids were washed with waterto form 1.42 Kg of wet cake containing: 283 g (23.44 mole) fumarate, 4.2g (0.031 mole) maleate, 8.8 g (0.074 mole) succinate, <9 g (<0.062 mole)alpha ketoglutarate and <3 g (<0.032 mole) glycerol.

The 1.42 Kg. wet cake was re-suspended in 1.5 Kg de-ionized water at 30°C. and 344 g of a 33% ammonia solution was added. Gaseous CO2 was thenbubbled through the suspension until the pH was 8.7. After cooling theambient temperature the suspension was filtered and the cake was washedwith 1.2 Kg. water. The wash water was combined with the filtrate. Thecomposition of the combined solution was 257 g (2.21 mole) fumarate(>90% conversion), 2.2 g. (0.016 mole) maleate, 8.2 g. (0.069 mole)succinate, 0.4 g (0.003 mole) alpha ketoglutarate 0.18 g (0.0045 mole)calcium and 90 g (5 mole) ammonia.

These results show that high conversion yields can be combined with asubstantial purification of the fumarate. The proportion of the fumaratein the total fermentation products increased from 67% in the broth to96% in the product ammonium fumarate.

It will be evident to those skilled in the art that the invention is notlimited to the details of the foregoing illustrative examples and thatthe present invention may be embodied in other specific forms withoutdeparting from the essential attributes thereof, and it is thereforedesired that the present embodiments and examples be considered in allrespects as illustrative and not restrictive, reference being made tothe appended claims, rather than to the foregoing description, and allchanges which come within the meaning and range of equivalency of theclaims are therefore intended to be embraced therein.

We claim:
 1. A process for the preparation of aspartic acid via a fermentation process for the preparation of ammonium fumarate, wherein the pH of the fermentation broth is controlled by the addition of a calcium base to produce a calcium fumarate precipitate, characterised in that ammonium fumarate is produced by separating said precipitated calcium fumarate from said fermentation broth, and reacting said precipitate with a reagent selected from ammonia, ammonium carbonate, ammonia in combination with CO₂ and mixtures thereof, to form ammonium fumarate solution and a co-product selected from calcium carbonate and calcium hydroxide, wherein the energy of indirect neutralisation of fumaric acid by ammonia serves as the driving force for the conversion of calcium fumarate to the desired ammonium fumarate product with a conversion yield of at least 90% and for the regeneration of a calcium base reagent, and wherein said ammonium fumarate product is enzymatically converted to ammonium aspartate and acidulated to form aspartic acid.
 2. A fermentation process according to claim 1, wherein said calcium base co-product is recycled to the fermentation broth.
 3. A fermentation process according to claim 1, wherein the amount of water introduced with said reagent is controlled such that it will not exceed the level equivalent to final fumarate concentration of about 50% of saturation.
 4. A fermentation process according to claim 3, wherein the amount of water introduced with said reagent is controlled such that the final fumarate concentration is at least 80% of saturation.
 5. A fermentation process according to claim 1, wherein said precipitated calcium fumarate from said fermentation broth is subjected to purification before reaction with said reagent.
 6. A fermentation process according to claim 5, wherein said precipitated calcium fumarate from said fermentation broth is recrystallized before reaction with said reagent.
 7. A fermentation process according to claim 1, wherein said precipitated calcium fumarate is first reacted with ammonia in an aqueous medium to raise the pH of the reaction medium to between about 10 and 11, and then ammonium carbonate is added thereto.
 8. A fermentation process according to claim 1, wherein the amount of CO₂ in said reagent is about 1 mole per mole of calcium fumarate.
 9. A fermentation process according to claim 1, wherein said reaction is carried out at a temperature below 80° C.
 10. A fermentation process according to claim 1, wherein said reaction is carried out at a temperature below 50° C.
 11. A fermentation process according to claim 1, wherein said reaction is carried out using gaseous CO₂ and is performed at a pressure below 5 atmospheres.
 12. A fermentation process according to claim 1, wherein said ammonium fumarate product is obtained at a purity of about 96% of the total carboxylates formed. 